TMT proteomics analysis of intestinal tissue from patients of irritable bowel syndrome with diarrhea: Implications for multiple nutrient ingestion abnormality.

Diarrheal irritable bowel syndrome (IBS-D) is a chronic functional bowel disease with no clear diagnostic markers and no satisfactory treatment strategies. In recent years, the importance of intestinal microstructure and function in IBS-D has been emphasized. However, the intestinal tissue proteomics of IBS-D patients has not been analyzed. Here, we systematically analyzed the molecule profiling of the intestinal tissues in IBS-D patients through tandem mass tag (TMT)-based proteomics for the first time, aiming to reveal the pathogenesis and provide evidence for diagnosis and treatment of IBS-D. Five IBS-D patients and five healthy subjects were selected, biopsy tissue samples from the junction of sigmoid and rectum were analyzed by TMT proteomics. Differentially expressed proteins were obtained and bioinformatics analysis was performed. Furthermore, parallel reaction monitoring (PRM) and q-PCR detection were applied to validate the differentially expressed proteins. Eighty differentially expressed proteins were screened, 48 of which were up-regulated and 32 were down-regulated (fold change >1.2, P < 0.05). Bioinformatics analysis showed that these proteins were significantly enriched in the nutrient ingestion pathways which are related to immune molecules. SELENBP1, VSIG2, HMGB1, DHCR7, BCAP31 and other molecules were significantly changed. Our study revealed the underlying mechanisms of IBS-D intestinal dysfunction. SIGNIFICANCE: Irritable bowel syndrome with diarrhea (IBS-D) is a worldwide chronic intestinal disease with no definite diagnostic markers. It is still a challenge to accurately locate the pathogenesis of patients for appropriate treatment strategy. Established proteomics studies of IBS-D are only based on urine, blood, or tissue samples from animals. Our study was the first TMT proteomics analysis on intestinal biopsy tissues of patients with IBS-D, which revealed the changes of molecular spectrum of actual intestinal conditions in patients with IBS-D. Some important molecules and signaling pathways have been found abnormal in our study, which were related with nutrient uptake. They not only provided preliminary clues for low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) intolerance, an unsolved conundrum of IBS-D, but also revealed obscure problems of protein, lipid, and other nutrients ingestion in IBS-D patients. Some of these differentially expressed molecules have been preliminarily verified, and will may be potential candidate molecules for diagnostic markers of IBS-D.

From Genotype to Phenotype: Content and Activities of Cytochromes P450 2A6 in Human Liver In Vitro and Predicted In Vivo.

Unraveling the molecular mechanisms by which genetic variants of cytochrome P450 2A6 lead to different metabolic phenotypes remains a long-standing but important challenge. CYP2A6 is an enzyme involved in the metabolism of several clinical drugs as well as the metabolic activation of carcinogenic nitrosamines. Herein, CYP2A6 genotypes and phenotypes, as indicated by protein content [by liquid chromatography-mass spectrometry (MS)/MS] and metabolic activities [Vmax, clearance (CL)], were determined for 90 human liver samples. We determined the median, range, and interindividual and intraindividual variation of CYP2A6 content and activity at the microsomal, liver tissue, and whole liver level and predicted hepatic in vivo clearance by in vitro-in vivo extrapolation based on CYP2A6-mediated coumarin metabolism by each CYP2A6 genotype. These results reveal how different CYP2A6 genotypes yield different phenotypic traits in protein content and enzyme activity. For relative Vmax, CL, and protein content, the intraindividual percentage coefficients of variation (ICVs) were 41.0% (18.8%-125.1%), 28.5% (2.39%-133.5%), and 27.8% (2.68%-88.0%), respectively. The high ICVs implied large intraindividual variation at different levels, sometimes in a genotype-dependent manner. Intergenotype analysis revealed that the CYP2A6*4 allele demonstrated the most obvious effect on phenotypic outcomes, both in protein content and in metabolic activity. Indeed, decreased CYP2A6 protein content with the CYP2A6*4 genotype might explain the decreased metabolic activity from the molecular to the organismal level. These findings may allow useful predictions for CYP2A6-mediated drug metabolism on an individual patient basis in accord with the goal of achieving personalized medicine. SIGNIFICANCE STATEMENT: We provide the median, range, and interindividual and intraindividual variation in CYP2A6 content at the microsomal, liver tissue, and whole liver level by liquid chromatography-mass spectrometry (MS)/MS as well as activities at the protein, microsomal, liver tissue, and whole liver level both in vitro and at the organismal level based on CYP2A6-mediated coumarin metabolism with each CYP2A6 genotype, thereby allowing us to elucidate how different CYP2A6 genotypes yield differing phenotypic traits (protein content and enzyme activity), facilitating the development of personalized medicine.

Intraindividual Variation and Correlation of Cytochrome P450 Activities in Human Liver Microsomes.

We systematically studied and explored intraindividual variation and correlation in the activities of cytochrome P450 (CYP) using 105 normal liver samples. The intraindividual percentage coefficients of variation of relative Km, Vmax, and CLint were 45.9% (18.3-140%), 44.0% (16.8-127%), and 52.0% (24.2-134%). Overall values of relative Km, Vmax, and CLint for 10 CYPs were sorted. The order, from highest to lowest, was CYP2D6, 3A4/5, 2C19, 2C9, 2B6, 1A2, 2A6, 2C8, and 2E1 for Km; CYP3A4/5, 2C19, 2D6, 2C8, 2E1, 2B6, 1A2, 2A6, and 2C9 for Vmax; and CYP2D6, 2B6, 3A4/5, 2C19, 2C9, 1A2, 2E1, 2C8, and 2A6 for CLint. CYP2A6*4, 2B6 785A>G, and 2D6 100C>T contributed to intraindividual variation of CYP activities. The correlations and similarities among 10 CYP activities were analyzed, and it was found that the highest correlation and similarity for Vmax, Km, and CLint occurred between CYP2C9 and 2C8, CYP2C9 and 1A2, and CYP2C9 and 2C8, respectively. In conclusion, CYP activities exhibit considerable intraindividual variation, with some notable correlations, which might be helpful to comprehensively understand the internal connection among CYP activities and to guide the rational use of drugs.

Prevalence of Spina Bifida Occulta and Its Relationship With Overactive Bladder in Middle-Aged and Elderly Chinese People.

PURPOSE: To investigate the prevalence of spina bifida occulta (SBO) and its relationship with the presence of overactive bladder (OAB) in middle-aged and elderly people in China. METHODS: A cross-sectional community-based survey was carried out at 7 communities in Zhengzhou City, China from December 15, 2013 to June 10, 2014, where residents aged over 40 years were randomly selected to participate. All of the participants underwent lumbosacral radiographic analysis and relevant laboratory tests. A questionnaire including basic information, past medical history and present illness, and the OAB symptom score was filled out by all participants. Chi-square tests and logistic regression were used for data analysis with a P-value of <0.05 denoting statistical significance. RESULTS: A total of 1,061 subjects were qualified for the final statistical analysis (58.8±11.7 years; male, 471 [44.4%]; female, 590 [55.6%]). The overall prevalence of SBO was 15.1% (160 of 1,061): 18.3% (86 of 471) in men and 12.5% (74 of 590) in women. Among these subjects, 13.7% (145 of 1,061) had OAB: 13.2% (62 of 471) in men and 14.1% (83 of 590) in women. The results of logistic regression showed that age, SBO, history of cerebral infarction (HCI), and constipation were risk factors for OAB (P<0.05), while sex, history of childhood enuresis (HCE), body mass index (BMI), and diabetes mellitus (DM) were not (P>0.05). In men, age, SBO, and constipation were risk factors for OAB (P<0.05), while HCE, BMI, DM, HCI, and benign prostate hyperplasia were not (P>0.05). In women, age, SBO, and HCI were risk factors for OAB (P<0.05), while HCE, BMI, DM, vaginal delivery, and constipation were not (P>0.05). CONCLUSIONS: The prevalence of SBO is high and it is related to OAB in middle-aged and elderly people in China.